Increased intrahepatic resistance to blood flow is an important component of many forms of portal hypertension. We and others have recently demonstrated that perisinusoidal stellate cells (Ito cells or lipocytes) exhibit a contractile phenotype and play an important role in regulation of sinusoidal blood flow. The dynamic nature of blood flow regulation within the hepatic sinusoid (or capillary in the systemic circulation) emphasizes the importance of interplay between vasoconstrictive and vasodilatory compounds. ET-1, a potent vasoactive peptide, is overproduced after liver injury and appears to contribute to increased intrahepatic resistance via contraction of stellate cells. In contrast, nitric oxide (NO), a key molecule in normal vascular homeostasis, has been identified as an important relaxing factor for stellate cells, and balances the effect of ET- 1. Available data therefore suggest that the balance of such vasoregulatory elements helps determine the degree of resistance within the hepatic microcirculatory unit. We have shown that after liver injury, NO synthesis is reduced. The abnormality has been localized to defective NO synthase (eNOS) dependent NO release by sinusoidal endothelial cells. Further, preliminary data presented in the current application have identified specific post-translational defects in eNOS after liver injury, including reduced phosphorylation of eNOS by the protein kinase, Akt. New data indicate that GRK2 interacts with and affects Akt activity. These data have led us to hypothesize that post-translational defects in eNOS after liver injury are critical in establishment of the sinusoidal endothelialopathy present after liver injury. The overall goal of the PI is to elucidate the cell and molecular basis of increased intrahepatic resistance with a focus on sinusoidal endothelial cells and eNOS. The specific aims of the current proposal are (1) to examine eNOS phosphorylation in the normal and injured liver, (2) to understand caveolin-1 expression and regulation in SECs, and (3) to investigate the role of GRK2 in regulation of Akt and eNOS function. The proposed experiments have important therapeutic implications not only for patients with liver disease and portal hypertension, but also across much of vascular biology. PUBLIC HEALTH RELEVANCE: Liver damage leads to elevated resistance to blood flow through the liver. This leads to intestinal bleeding and severe clinical consequences. This proposal aims to understand the cause of elevated resistance to blood flow and would shed light on potential treatments for this problem.